Sickle cell anemia is accompanied by biochemical activation of the coagulation system and by clinical thromboses. Therefore, we have focused on the expression of tissue factor (TF), the trigger of the coagulation system, in this disease. Previous studies from our laboratories described abnormal TF expression by monocytes and by circulating endothelial cells in human sickle blood. More recently, we reported (
) that severe-phenotype (BERK) sickle mice abnormally express TF on pulmonary vein endothelium; mild-phenotype (NY1DD) sickle mice are normal in this regard but assume the abnormal phenotype after exposure to hypoxia (3 hr at 8% O2) followed by reoxygenation for 18 hr at ambient air (H/R). Monitoring the % of pulmonary veins positive for TF on triple stained tissue sections (for nuclei, for murine TF, for murine CD31 to identify endothelial cells), we have now evaluated the role of NO in TF expression, employing these two models. In the NY1DD H/R model, NO inhalation (34 ppm) administered during the entire reoxygenation period led to 68±18% reduction (P<.001) of TF expression. Consistent with this, dietary supplementation with arginine (substrate for nitric oxide synthase [NOS]) prior to H/R led to 93±25% reduction (P<.001) of the H/R induced TF response. Conversely, dietary supplementation with L-NAME (inhibitor of NOS) converted the NY1DD mouse to a TF positive phenotype (P=.047) without even requiring H/R. Interestingly, inhalation of NO was not protective in the NY1DD H/R model if given only during the hypoxia period itself or only during the last 3 hours of the reoxygenation period, but it was protective if given only during the first 3 hours of the reoxgygenation period (68±24% reduction; P=.0008). Thus, the timing of clinical intervention is probably critical. In the BERK model at ambient air, treatment with dietary arginine for 2 weeks diminished TF expression by 60±33% (P=.04), an effect that was countervailed by concurrent administration of L-NAME. Data will soon be available on eNOS over-expressing sickle mice. Also in BERK mice, L-NAME largely prevented the TF inhibiting therapeutic benefit of lovastatin, suggesting that lovastatin’s beneficial effect on TF is mediated via NO biology. These results suggest that NO biology determines endothelial TF expression in vivo in sickle mice. We note that NO is reported to suppress endothelial TF expression in vitro. We find that sickle mice have elevated plasma free Hb levels (increased by 40% in NY1DD mice and 57% in BERK mice), a risk factor for diminished NO bioavailability. On the other hand, H/R in NY1DD mice is not associated with a further increase in free plasma Hb. So it seems that the NO biology of sickle mice is not simply explained by plasma Hb. NO biology seems to exert a major role in regulating endothelial TF expression in the sickle mouse. Long-term NO modulating therapy, such as dietary arginine or lovastatin, may be beneficial in terms of the coagulopathy and thrombosis risk of sickle patients and should be tested with this in mind.
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